Camille Stanback
Identifying an Intraflagellar Transport Protein: Fla15p in Chlamydomonas
Mentor:  Dr. Susan Dutcher, Department of Genetics, Washington University School of Medicine 

Chlamydomonas reinhardtii is a unicellular eukaryotic green alga.  This biflagellate organism serves as an excellent model for the analysis of human ciliary diseases because of the ease of genetic, biochemical, and cell biological manipulations.  Cilia or flagella, are microtubule based projections encased in membranes.  Motile cilia propel cells through liquid while sensory cilia detect signals in the environment.  

Intraflagellar transport (IFT) is the process by which a large multimeric protein complex is moved along flagella by molecular motors.  It carries flagellar building blocks to the tip of the flagella and returns signals to the cell body and recycles used components.  I studied fla15-1, a temperature-sensitive mutant of Chlamydomonas.  At the restrictive temperature, mutant cells lack flagella.  At the permissive temperature, the rate of inward IFT movement is reduced compared to wild-type cells.  Biochemically, one of the two subcomplexes of IFT, Complex A, is reduced.  Previouswork in the lab showed linkage between  the IFT144 and FLA15 genes.  Ift144p is a component of Complex A (Iomini, Cole, and Dutcher personal communication) and they also showed that a C-terminal cysteine  is changed to an arginine in the fla15-1 strain.  In my project, I used reversion analysis to provide evidence that this change is responsible for the mutant phenotypes of the fla15-1 strain.

Following ultraviolet mutagenesis of fla15-1 cells, cells with a wild–type phenotype at the restrictive temperature were isolated.  27/27 strains examined changed the DNA sequence of IFT144 around cys.  This result suggests that IFT144 is the gene product of FLA15.  These data rely on a Restriction Fragment Linked Polymorphism, which leave some ambiguity in the interpretation.  If time had permitted, I would have sequenced several of the revertants to determine if I found true revertants (CGC - TGC) as opposed to pseudorevertants (CGC - GGC).